Post-ischemic neovascularization:

basic concept and new treatment options

Professor Costanza Emanueli, PhD

British Heart Foundation Senior Research Fellow

Professor and Chair of Vascular Pathology and Regeneration- Bristol

Visiting Professor -ICL

ESC Summer school, Nice 16 June 2013

Index of this Lecture

•Introduction to ischemia and post-ischemic neovascularisation

•Research Models

• Therapeutic angiogenesis research: past, present and future

•microRNAs in post-ischaemic vascular regeneration

•Novel stem cell products: Empowered EPCs, Pericyte progenitor cells

Ischemic Disease

•Tissue ischaemia is caused by poor blood flow supply, which impairs the delivery of oxygen (creating hypoxia) and nutrients (creating tissue starvation)

•Ischaemic disease is favoured by atherosclerosis, diabetes, aging and a series of cardiovascular risk factors

• Ischaemic disease is one of the biggest medical epidemic worldwide and its treatment is still an umet clinical need.

Post-Ischaemic Vascular Regeneration: the Medical Need

MYOCARDIAL INFARCT

ISCHAEMIC STROKE

CRITICAL LIMB

ISCHAEMIA

LIMB ISCHAEMIA

Vascular Regeneration: what can we try?

Substituting the diseased (large) vessel

By-pass grafting with pieces of patient own vessels (vena saphena and/or mammary artery)

By-pass grafting with “artificial” vesselscreated in the laboratory

Therapeutic Neovascularization

VASCULOGENESISischemiaischemia

ischemia

SDF-1,

VEGF, IL-8, ....

SDF-1,

VEGF, IL-8, ....

Strategies for Post-Ischemic Neovascularisation

• Increase the expression/activity of endogenous “pro-angiogenic” factors in ischemic tissues

• Stimulate endogenous stem and progenitor cells with various pro-angiogenic capacities

• Transplantation of vascular stem and progenitor cells with pro-angiogenic capacities

• Contrast “anti-angiogenic” molecular signatures induced by pathology and/or risks factors

In Vivo Research Models

Occlusion of LAD

Experimental models of myocardial infarction (MI) are routinely performed in Rodents.- Permanent ligation of LAD- Ischemia/reperfusion

Animal Models of Ischemia: MI

Mouse model of myocardial infarct

Ligation of the (left) femoral

artery

Mouse ventral view

Experimental models of limb ischemia (LI) is used in mice to mimic a situation comparable to peripheral artery disease. Since peripheral artery disease is often associated with diabetes, this model is also performed in diabetic mice. LI is performed by permanent ligation of one femoral artery.

Animal Models of Ischemia: LI

Intravenous infusion of stem cells/genes:

through the femoral vein

through the tail vein

through the left ventricular cavity

Limits of systemic delivery:

- Not possible to target a specific organ- (Loss of cell number)- (Loss of cell viability)

Systemic Gene/Cell Delivery

Cardiac:

1. Endomyocardial needle injection

2. Catheter delivery (large animals)

3. Intracoronary infusion (large animals/humans)

4. Intrapericardial (large animals)

Lower limb

1. Direct intramuscular injection

2. Intra-arterial infusion

Cells/Genes are directly injected into the infarcted border zone

Injection is often performed immediately after MI

Cells/genes are directly injected into the adductor muscle often immediately after ischemia

Local Gene/Cell Delivery

Body distribution of cells labelled with D-luciferin after intracardiac injection of different types of stem cells

Time after cell injection

Types of injected stem cells

BM-derived mononuclear cells

Skeletal myoblasts

Mesenchymal stem cells

Fibroblasts

Van der Bogt et al, Circulation 2008

In vivo optical bioluminescence imaging (by using luminescence protein)

Transgene/ Cell Tracking after Local Delivery

New 3D bioluminescent IVIS system (Caliper)

Non Invasive Colour Laser Doppler

Blood flow ratio (ischemic/contralateral foot)

Blood Flow Recovery

GroupA

Group B

Histology /IHC for vessel analyzes

NGF

Cap

/mm

2

Small Arteriole densitySmall Arteriole density

2

†

0204060

10080

140120 **

**††

**

6000**††

5500

4000

45005000

3500

3000

***††

6000**††

5500

4000

45005000

3500

3000

*

30 days

††

14 days

MI/Ad.Null

MI/Ad.Null

MI/Ad.hNGF

00.10.20.30.40.50.60.70.80.9

****

†

00.10.20.30.40.50.60.70.80.9

(ml/m

in/g

)

****

†

30 days 14 days 30 days

MyocardialMyocardialBlood FlowBlood FlowA

rt/m

m2

Capillary densityCapillary density

Sham/NullSham/NullMI/NullMI/NullMI/NGFMI/NGF

14 days

IsolectinB4/CD45/BrdU

Blo

od F

low

to L

V

(mL

min

-1 g

r-1)

2

4

6

8

0Ad.Null Ad.NGFSham

*§§

§§

Artificial Respiration

Microsphere injection in LV.After 2 minutes, collect tissues

Reference Blood FlowCollection (170ml/min)

BF (mL/min/gr) R x (fl tissues/ fl ref blood) gr of tissue

=

R = rate (mL/min) of ref blood withdrawal

Absolute Blood Flow Measurement in Tissues

MI

Meloni et al. Circ Res, 2010

Mouse Echocardiography

Day 0Day -7

Arrival

From

Farm

Myocardial

Infarction

(n=45)

Cath lab

Day 14 Day 15

Surgical injection

Randomization to**:

- Group A (n=13)

- Group B (n=13)

- Group C (n=13)

BASELINE

IMAGING

Day 28

intermediate

IMAGING

Day 56

FINAL IMAGING

(endpoint)

*Randomization will

be based on baseline

LVEF by MRI.

From Dr Borja Ibanez, CNIC-Madrid

Large animal models for translational research

Spontaneous post-ischemic neovascularization process

Copyright ©1999 American Heart Association

OLDRABBIT

YOUNG RABBIT

Aging impairs post-ischemic collateral formation and healing in limbs

MICE

Experimentallimb ischemia

Rivard & Isner, Circulation, 1999

Van Belle, & Isner Circulation 1997

Hypercholesterolemia attenuates post-ischemic angiogenesis in limbs

NORMALRABBIT

ATHEROSCLEROTICRABBIT

diabetic

diabetic

Diabetes impairs post-ischamic angiogenesis and blood flow recovery

Foot Blood Flow Recovery

Control mouse

Diabetic mouse

Rivard & Isner, Am J Pathol, 1999

Emanueli & Madeddu. Hypertension 2001

High blood pressure impairs post-ischemic angiogenesis and blood flow recovery in rats

hypertensivehypertensive

No-ischemia

Ischemia

Foot Blood Flow RecoveryCapillary Density

Ischemic patients have at least one of these aforementionedconditions and often all of them plus additional risk factors(obesity, “wrong” genetic and epigenetic backgrounds, smoke,

etc) to prevent spontaneous healing.

They need some help to fight ischaemia!

MYOCARDIAL MYOCARDIAL INFARCTINFARCT

ISCHAEMIC ISCHAEMIC STROKESTROKE

CRITICAL CRITICAL LIMB LIMB

ISCHAEMIAISCHAEMIA

LIMB LIMB ISCHAEMIAISCHAEMIA

Therapeutic Neovascularisation ofCardiac and Peripheral Ischaemia

Jeff Isner

First Stretegic Mind to use Angiogenesis as a Therapy

• First attemptTo increase the expression of the prototypicalpro-angiogenic factor VEGF-A in ischaemic tissues using asupply-side approach, mainly by gene therapy viaplasmids and first generation adenoviruses

• Second attemptTransplantation of endothelial progenitor cells (EPCs) in ischaemic tissues.Done in collaboration with Prof Asahara then a fellow of Isner. This approach

was then clinically translated by others (mainly in Frankfurt and Japan)

“Classical” pro-angiogenic factors in mono-therapies

Vascular stabilisation vs vascular regression

Options for Improving Traditional Post-Ischaemic Neovascularisation Therapeutics

• More basic science to understand all the players in the angiogenesis and anti-angiogensis orchestras

•Test different angiogenesis modulator factors (growth factors and more)

•Combinatory approaches

•More severity in preclinical testing (large animal models, risk factors associated to experimental ischemia, etc)

•Work at gene vectors

•Success in in vivo small vessel imaging

•Design better clinical trials

•Do not give up!

microRNAs (miRs)

DicerThomas ThumBreak-out sessionTuesday 2PM

Dicer generates short interfering RNAs (including miRNAs) from longer double-stranded RNAs.

Mice homozygous for a hypomorphic allele of Dicer showed impaired developmental angiogenesis and died at days 12.5 - 14.5 of gestation

Yang W J et al. J. Biol. Chem. 2005;280:9330-9335

First evidence for a regulatory role of miRs in vascular development

LowDicer

Spontaneous post-ischaemic neovascularisation

process

VASCULOGENESIS ischemiaischemia

ischemia

SDF-1,

VEGF, IL-8, ....

SDF-1,

VEGF, IL-8, ....

VASCULOGENESIS

ANGIOGENESIS

Spontaneous post-ischemic neovascularization process

Scientific Questions:

Do miRs modulate it? What miRs? At what levels?

Therapeutic exploitation of miRs?

Bone marrow-derived endothelial progenitor cells (EPCs)/proangiogenic circulating cells (PACs)

Mouse limb ischaemia model

Grundmann & Moser, Circ , 2011

Hypoxia

Kulshreshtha &, Ivan, CDD, 2008.

miR expression is regulated by hypoxia and ischemia

Systemic miR-92a inhibition stimulates recovery after hind limb ischaemia

Day 0,2,4,7, and 9Antagomir 92a or Co

Hind limb ischemia

Blood flowMicrovessel density

Antagomir-Co

nuclei Lectin

SMA merge

Antagomir-92a

nuclei Lectin

SMA merge

Ischemic leg Ischemic leg

Antagomir-Co Antagomir-92a

Slide kindly provided by Stefanie Dimmeler

Bonauer& Dimmeler, Science, 2009

miR-92a: first example of mIR targeting for post-ischemic angiogenesis

Example for miR targeting and contrasting anti-angiogenic factors

2011

miR-503: how we joined the miR club!

Andrea Caporali

Finnerty JR et al. JMB 2010

Member of miR-15/107 group: AGCAGC seed sequence

Chr X; intergenicCluster: miR-424-503

Examination of the mir-424 and mir-503 locishowed that they are separated by 383 bases onthe genome and derived from the same primarytranscript

miR-503

NG

0

1

2

3

4

5

miR

-503

rela

tive

expr

essi

onHG

LGF

HG-LGF

*

miR-503 expression in HUVECs

miR-503 expression in ECs is increased by culture conditions mimicking hyperglycaemia and ischemia

NG HG

LGF HG-LGF

NG= normal glucose, 5 mM

HG= high glucose, 25 mM

LGF = reduced growth factors

0

20

40

60

80

100

G0/G1 S G2/M

miR503GFP

% c

ells

gat

ed

*

*0

20

40

60

80

100

G0/G1 S G2/M

miR503GFPmiR503GFP

% c

ells

gat

ed

*

*

GFP miR503miR503GFP

Impaired EC migrationImpaired EC network formation

Impaired EC cycle miR-503 target genes

Consequences of miR-503 overexpression in endothelial cells

Local gene therapy with a decoy for miR-503 improves post-ischaemic angiogenesis in diabetic mice

0

2

4

6

miR

-503

rela

tive

expr

essi

on

++#

Non diabetic Diabetic

IschNo Isch IschNo Isch

††

miR

-503

rela

tive

expr

essi

on

IschNon Diab

0

1

2

3

IschDiab

CD146pos ECs

†

Adductor Muscle

1

3

5

Foot

BF

Rec

over

y

30 min 7d 14d0.0

0.2

0.4

0.6

0.8

Time post-ischemia21d

***

§ §§

decoy503Null

Non Diab Isch Diab Isch Diab Isch

1.0

miR-503 expression

Non Diab Isch + Null Diab Isch + decoy503Diab Isch + Null

Limb microvessels

Blood flow recovery

New cell therapy products

# E

ven

ts

Fluorescence intensity

SS

C-A

FSC-A

PACs

99%

AcLDLUEAI

98%#

Even

ts

Fluorescence intensity

Negativecontrol

Specific marker

KDR

59±7%

CD34

11±2%

CXCR4

68±8%

CD45

82±5% 88±8% 90±6%

CD11bCD14

Phenotypic characterization

4 DAYS CULTURE IN EC MEDIUM

ADHERENCE ENRICHMENTON FIBRONECTIN

HISTOPAQUE MNC SEPARATION

Plasma

MNCs

erythrocytesFLOATING

ADHERENT CELLSIsolation and culture

PACs

Human circulating pro-angiogenic cells: PACs

Circ Res, 2013

Gaia Spinetti

miR screening in blood PACs of CLI Patients with/out Diabetes

PAC miR-15a and -16 expression can be manipulated ex vivo

Pre-miR Anti-miR

miR-15a and -16 overexpression increasesapoptosis of “healthy” PACs

miR-15a and -16 inhibition improves survival of patient-derived PACs

Overexpressing miR-15a and miR-16 inhibitsthe migratory capacity of healthy PACs

Inhibition of miR-15a together with miR-16improves the migratory capacity of diseased PACs

miR-15a and miR-16 target VEGF-A and AKT-3

3’UTR luciferase activity assays

Ex-vivo miR-15a and miR-16 inhibition in PACs increases their regenerative potential:

PACs+SCR

PACs+pre-miR15a/16 PACs+anti-miR-15a/16

Medium

Foot Blood flow....

.... Microvascular Density

Spinetti G et al. Circ Res, 2013

Blood circulatingmiR-15a and -16

CLI patients with DM undergoing PTA

Association between serum miR-15 or miR-16 at revascularization and restenosis/amputation at follow up (1 year)

miRNA Summary and Perspective

•miRs modulate the post-ischemic neovascularization responses at multiple levels

•miR therapeutic can directly target ischemic tissues in vivo and be used for ex-vivo enhancement of the proangiogenic capacities of stem and progenitor cells

•Circulating miRs might be novel predictive and pognostic markers in ischemic patients

•There is a huge potential for miR translational research

Bristol Vena Saphena derived-Pericyte Progenitor Cells (SVPs)

Paolo Madeddu-Wednesday AM

Isolectin/NG2

2 weeks post-ischemia

Limb muscle, basal conditions

Pericyte coverage of is essential for microvessel maturation and stabilisation, including during post-ischemic vascular repair

NG2pos pericytes around neovessels

59

Isolectin/NG2

The saphenous vein: a convenient source of pericyte progenitor cells

Vein leftover

60

CABGCampagnolo et al, Circulation, 2010

CD34 vWF

DAPI CD34 vWF DAPICD34 vWF DAPI

DiDii Diii

Div Dv

E F

CD34 vWF

DAPI CD34 vWF DAPI

Dii Diii

Div Dv

Campagnolo et al. Circulation 2010

CD34 vWF

DAPI CD34 vWF DAPICD34 vWF DAPI

DiDii Diii

Div Dv

E F

Tunica media

Adventitia

Vasa vasorum

Identification of pericytes in human saphenous vein vasavasorum

61

CD34vWFDAPI

CD34

CD34 CD31NG2 DAPI

CD34 CD31PDGF-R bDAPI

vWF

CD34 vWF DAPI

Conclusions Myocardial and limb ischaemia are still unmet medical

needs

Novel options and revisited approaches hold therapeutic potential to treat limb and myocardial ischaemia

This area of research can deliver disappointments as well as huge satisfaction

Basic science and translational approaches must progress together to the ultimate clinical goal.

Main collaborators

Emanueli Lab BristolMarco MeloniBetty DescampsTijana MiticLynsey HowardSaran ShantikumarIlaria FlorisMicol MarchettiSobia MushtaqAnran Li

Andrea CaporaliAudrey Nailor

Angelini & Emanueli Lab ImperialAbas H LaftahMore to be appointed...

BristolPaolo Madeddu & LabAndrew MumfordRaimondo AscioneChiara Bucciarelli-DucciGianni AngeliniGraciela Sala-NewbyBarney Reeves BRU unit

ImperialSian HardingAnna RandiMark Perry

GlasgowAndy BakerAshley Miller

KCLQingbo Xu

EdinburghDavid NewbyBruno PeaultRoslin Cells

Sheffield Tim Chico

MilanGaia SpinettiGianluigi Condorelli Maurizio CapogrossiDiego PasiniFabio Martelli

SassariGianfranco Pintus

UdineAntonio Beltrami

HannoverThomas Thum

FrankfurtStefanie DimmelerCarlo Gaetano

YaleBill SessaCarlos Fernandez

UmassNathan Lawson

MadridIntegromics

ViennaKlemens Verlienger

Emanueli & Madeddu Labs in Bristol

We are currently recruiting and always happy to host visiting PhD students and sponsor excellent candidates for

fellowship applicationscostanza.emanueli@bristol.ac.ukmadeddu@yahoo.com